The spread of a newly evolved coronavirus (CoV) caused a global threat of severe acute respiratory syndrome (SARS) pandemics in 2003 (Kuiken, T. et al., 2003, Lancet 362: 263-270). Coronaviruses are taxonomically classified in the order Nidovirales, based on features of their genome organization and replication strategy. As with other coronaviruses, SARS-CoV has the morphology of enveloped particles with typical peripheral projections, termed “corona” or “spikes,” surrounding the surface of a viral core (Ksiazek, T. G. et al., 2003, N Engl J Med 348: 1953-1966; Lin, Y. et al., 2004, Antivir Ther 9: 287-289). Outside the coronavirus particle core is a layer of lipid envelope containing mainly three membrane proteins, the most abundant M (membrane) protein, the small E (envelope) protein, and the S (spike) protein. The homo-trimers of the S protein collectively form the aforementioned corona, which is involved in viral binding to host receptors, membrane fusion for viral entry, cell-to-cell spread and tissue tropism of coronaviruses. The viral core inside the envelope, termed nucleocapsid, harbors a positive-strand viral genome RNA of approximately 30 kb packaged by the N (nucleocapsid) protein.
Unlike other human coronaviruses, such as HCoV-229E and HCoV-OC43, that can cause only symptoms like the common cold, SARS-CoV causes a highly transmittable, severe and virulent disease that can often be lethal in adults and especially the elderly. Research and clinical interest on SARS-CoV has grown rapidly owing to the high infectivity and mortality. There is especially an urgent need for an effective and safe vaccine against SARS-CoV to deal with possible future reemergence of the SARS epidemics.
Most antiviral vaccines currently in use contain whole viruses, either inactivated or live-attenuated. Inactivated, or killed, viruses are treated chemically or by irradiation to disable their replication and are generally safe and easy to make. While eliciting neutralizing antibodies, they are unlikely to deliver viral antigens to cytosol for cytotoxic CD8+ T lymphocytes (CTLs) activation, which is critical to defend animals from infection. Live-attenuated vaccines are significantly more potent than killed vaccines. However, live-attenuated viruses pose the risk of reversion or recombination with circulating wild type into a virulent strain. Moreover, the manufacture of vaccines based on whole viruses also carries the risk of viral escape.
To avoid the danger of using the whole virus (such as killed or attenuated viruses) as a vaccine, recombinant viral proteins have been pursued not only as research tools but also as potential advanced subunit vaccines. However, subunit vaccines are known to suffer often from poor immunogenicity, owing to incorrect folding, poor antigen presentation, or difference in carbohydrate and lipid composition. Virus-like particles (VLPs) are self-assembled microscopic antigenic structures that resemble a virus in size and shape but lack genetic materials. VLPs can concurrently present viral proteins, carbohydrates and lipids in a similar and authentic conformation and thus have been viewed as an ideal vaccine against viruses (McGuigan, L. C. et al., 1993, Vaccine 11: 675-678). VLPs display intact viral antigens on the surface in a repeated arrangement, with which they afford the natural binding of a large viral entity to membrane receptors of antigen-presenting cells (APCs), such as dendritic cells (DCs). DC-targeted uptake of VLPs enables potent stimulation of CD4+ T cells against VLP-associated antigens. Besides stimulating humoral immunity, VLPs are permissive for cross-presentation in DCs that allows priming of CTL response with VLP-associated antigens (Moron, G. et al., 2002, J Exp Med 195: 1233-45).
VLPs for over thirty different viruses have been generated in insect and mammalian systems for vaccine purpose (Noad, R. and Roy, P., 2003, Trends Microbiol 11: 438-44). It has been shown that cellular expression of the M protein accompanied by the E protein of coronaviruses was a minimal requirement and sufficient for the assembly of VLPs (Vennema, H. et al., 1996, EMBO J. 15: 2020-2028). While being dispensable in forming VLPs, the S protein can be integrated into the VLPs whenever available (Godeke, G. J. et al., 2000, J Virol 74: 1566-1571).
Researchers have used baculovirus expression systems to produce SARS VLPs (Ho, Y. et al., 2004, Biochem Biophys Res Commun 318: 833-838; Mortola, E. and Roy, P., 2004, FEBS Lett 576: 174-178). However, due to the intrinsic differences between insect cells and mammalian cells, the VLPs assembled in the insect (SF9) cells exhibited a size of 110 nm in diameter, which is much larger than the 78 nm of the authentic SARS-CoV virions (Lin, Y. et al., 2004, supra, and Ho, Y. et al., 2004, supra). Moreover, immunogenicity of the insect cell-based SARS-VLP remains uninvestigated. Other researchers also tried to use mammalian expression systems to produce SARS VLPs (Huang, Y. et al., 2004, J Virol 78: 12557-65). However, the extracellular release of VLPs is not efficient, and the yield of VLPs is not satisfying.
Therefore, there is still a need for an efficient method for the large-scale production of SARS VLPs in order to provide an effective and safe vaccine against SARS.
Influenza infection is a major threat to human health and results in significant morbidity and mortality worldwide. According to World Health Organization estimates, seasonal influenza epidemics influence 5˜15% of the global populations annually and are responsible for more than 3-5 million hospitalizations and about 250,000 to 500,000 deaths per year (www.who.int/mediacentre/factsheets/fs211/en/index.html). Recently, in addition to the yearly circulating seasonal influenza variants caused by antigenic drift, other influenza virus strains with pandemic potential such as the highly pathogenic avian H5N1 or emerged novel A/H1N1 pose greater threats than in the past (www.who.int/csr/disease/avian_influenza/country/en/, and www. who.int/csr/don/2009—08—19/en/index.html) since they have become better adapted to humans by reassortment. The most efficient way of reducing the transmission of and the subsequent huge economic loss caused by seasonal or pandemic outbreaks of influenza is preventive vaccination. The manufacture of the current licensed influenza vaccines, either in the form of a split subvirion (disrupted, highly purified virus) or a subunit vaccine (purified hemagglutinin, HA, and neuraminidase, NA), is absolutely dependent on fertilized chicken eggs as a production bioreactor. This method has substantial limitations since the manufacturing capacity is restricted by the availability of eggs, which may be insufficient to meet the urgent requirements for vaccine during a pandemic [1,2,3]. In addition, these vaccines induce antibodies primarily to the viral HA and are efficacious in healthy adults, but display lower protective rates in high-risk groups (e.g., the elderly) and may be poorly immunogenic in young children. These problems are compounded once the wild population of virus undergoes significant antigenic drift in the HA component [1,2,4,5,6]. Consequently, the protective immunity elicited by inactivated vaccines is of too short a duration to protect from newly developed influenza variants. Therefore, the development of vaccines with cross-protective efficacy to allow a rapid response to influenza evolution and/or to prolong the efficacy of vaccination needs to be addressed.
Alternatively, an improvement in the preparation of seasonal influenza vaccines licensed in Europe uses reverse genetics in mammalian cell-based culture systems rather than in eggs [2]. Using mammalian cell culture systems such as Vero or MDCK cells as adaptive hosts for vaccine viruses has several advantages, not only increasing the flexibility and consistency of the manufacture process but also recovering the host-dependent specific glycosylation of viral antigens which may not be glycosylated properly in egg- or baculovirus-dependent systems. In eukaryotic cells, protein glycosylation is involved in correct folding or directing the cellular localization of newly translated proteins and plays important roles in protein function. Different glycosylation patterns underlie some of the differences between various strains of the influenza virus.
Recently, the use of noninfectious virus-like particles (VLPs) that self-assemble by spontaneous interactions of viral structural proteins has been considered to offer good potential for advanced vaccines for a wide range of viruses that cause disease in humans [7]. The VLP-based vaccine approach is an attractive alternative to replace or complement the conventional inactivated virus vaccines or subunit vaccines with improved safety and efficacy, especially for children and the elderly. It is worth noting that a VLP-based human papillomavirus (HPV) vaccine produced in yeast system which is capable of inducing protective immune response against the HPV responsible for cervical cancer was approved for the market in 2006 [8, 27, 28]. Influenza VLPs expressed by recombinant baculovirus systems that present multi-component antigens, including HA and matrix 1 (M1), with or without NA, and that are capable of inducing cognate or innate immune responses against homologous or heterologous strains of influenza virus, have been described [3,9,10,11,12,13,14, 29]. Clinical studies for baculovirus-expressed influenza VLPs are currently being undertaken.
In light of the great threats posed by seasonal and pandemic influenza infection, there is a need for further improved means for the development of flexible, effective, and safe vaccine for influenza infection.